Molecular Mechanism Of Dihydromyricetin In Diabetic Neuropathic Pain With Depression

Background:Diabetes mellitus（DM）is a chronic disease with a high incidence,which is mainly characterized by hyperglycemia in patients.The chronically high blood sugar in DM patients leads to chronic damage to various tissues of the body,and there is currently no effective method to cure DM.The clinical treatment for DM is mainly based on glycemic control,but currently many DM patients in china have poor glycemic control,so DM patients have a variety of complications,of which diabetic neuropathic pain（DNP）and depression（DP）are common complications.The comorbidity of the two has brought more serious physical and mental effects to patients with DM and is more difficult to treat.Therefore,there is an urgent need for more effective treatments.The P2X₇ receptor is one of the members of the purinergic receptor family and is widely divided in astrocytes and microglia in the nervous system.Dihydromyricetin（DHM）has various pharmacological effects such as anti-inflammatory and antioxidant.Our previous studies have shown that DHM can alleviate the behavior of DNP and DP in rats by reducing the activation of glial cells and the expression of P2X₇ receptor in DNP and DP combined rats.Brain-derived neurotrophic factor（BDNF）is an important member of the neurotrophic family,and it is closely related to the pathogenesis of depression and pain.Therefore,we speculate that BDNF also plays an important role in DNP and DP comorbidities.Therefore,this study further observed the effect and mechanism of DHM on BDNF levels in DNP and DP combined rats,and further observed the effect and mechanism of DHM on P2X₇ receptor at the cellular level.To provide an experimental basis for clinical treatment of DNP and DP comorbidities.Part OneObjective:In this study,the establishment of DNP and DP combined rat model was used to observe the effect of DHM on BDNF and Trk B in Hippocampus,SPI and DRG of combined rats and its mechanism of action,so as to provide a new method for clinical treatment of both comorbidities.Methods:Male SD rats were randomly divided into 4 groups:（1）control group（Control）,（2）control+dihydromyricetin group（Control+DHM）,（3）model group（DNP+DP）,（4）model+dihydromyricetin group（DNP+DP+DHM）.After the model was established successfully,the behavioral changes of the model rats were monitored through the thermal withdrawl lateney（TWL）,mechanical withdrawal threshold（MWT）,forced swimming test（FST）,sucrose preference test（SPT）and open-field test（OFT）.Western blot（WB）and Quantitative Real-time PCR（q PCR）were used to detect the expression levels of BDNF、Trk B、IL-1βand TNF-αin the hippocampus,spinal cord and DRG of rats.The expression of BDNF and Trk B receptor and neuron marker Neu N in rat hippocampus,spinal cord and DRG were detected by immunofluorescence.Results:1.After successful establishment of the rat model,compared with the control group,the behavioral test TWL,MWT,SPT and OFT of the model group were significantly reduced,and the FST immobility time was significantly increased（p<0.01）.After DHM treatment,TWL,MWT,SPT and OFT in the DHM treatment group were significantly higher than those in the model group,and FST immobility time was significantly reduced（p<0.01）.2.The results of q PCR and Western blot showed that in DRG and spinal cord tissues,the m RNA and protein expression levels of BDNF and Trk B in the model group were higher than those in the control group,while the m RNA and protein expression levels of BDNF and Trk B in the hippocampus were lower than those in the control group（p<0.01）.DHM treatment can significantly reverse these changes（p<0.01）.In hippocampus,DRG and spinal cord tissues,the IL-1βand TNF-αm RNA and protein expression levels of the model group were higher than those of the control group.However,the m RNA and protein expression of IL-1βand TNF-αin the DHM treatment group was lower than that in the model group（p<0.01）.3.Immunofluorescence results showed that BDNF,Trk B and Neu N immunofluorescence double-labeling showed that BDNF-Neu N and Trk B-Neu N were co-expressed in hippocampus,DRG and spinal cord tissue,and BDNF and Trk B were mainly expressed in DRG neurons.The co-expression of BDNF-Neu N and Trk B-Neu N in the spinal cord and DRG of the model group was higher than that of the control group,and DHM treatment can reduce this increase（p<0.01）.In the hippocampus,the co-expression of BDNF-Neu N and Trk B-Neu N in the model group was lower than that in the control group,while DHM treatment reversed this decrease（p<0.01）.Conclusion:DHM can reduce the neuropathic pain and depressive behavior of diabetic rats by regulating the expression level of BDNF in the nervous system.The mechanism may involve the inhibition of the release of inflammatory factors IL-1βand TNF-α.DHM is expected to become a effective drug for clinical treatment of DNP combined with DP.Part TwoObjective:The purpose of this study is to observe whether dihydromyricetin（DHM）has a protective effect on primary hippocampal astrocytes damaged by high glucose（HG）,substance P（SP）and corticosterone（CORT）and its possible mechanism.Methods:Primary culture of SD suckling rat hippocampal astrocytes was carried out by primary culture method.We randomly divided the purified primary astrocytes into 8 groups:control group（Control）,control+dihydromyricetin group（Control+DHM）,control+solvent group（Control+Solvent）,control+transfection reagent group（Control+Transfection reagent groups）,high glucose+substance P+corticosterone group（HG+SP+CORT）,high glucose+substance P+corticosterone+dihydromyricetin group（HG+SP+CORT+DHM）,High glucose+substance+corticosterone+P2X₇ sh RNA（HG+SP+CORT+P2X₇ sh RNA）and high glucose+substance+corticosterone+scramble sh RNA group（HG+SP+CORT+scramble sh RNA）.Through CCK-8 and ELISA,the cell viability was detected and the drug stimulation concentration was screened.After the cell model was successfully established,we detected the expression of P2X₇ receptor m RNA and protein in primary hippocampal astrocytes by q PCR and Western blot.Immunofluorescence double labeling method was used to detect the expression of P2X₇ receptor in cells.The expression levels of IL-1βand TNF-αin primary hippocampal astrocytes were detected by q PCR,Western blot and ELISA.The phosphorylation of ERK in each group of cells was detected by Western blot.Fluo-3 AM kit was used to detect the release of Ca²⁺signal in each group of primary hippocampal astrocytes.Flow cytometry was used to observe the apoptosis of primary cultured astrocytes in each group.Results:1.Purity identification result:primary cells cultured with GFAP and DAPI,immunofluorescence results show that the purity of primary cultured hippocampal astrocytes is>95%.2.Modeling concentration screening:According to literature research and preliminary experiments,it was determined that the concentration of HG was 30 m M,the concentration of SP was 5n M,the concentration of CORT was 0.1 m M,the optimal concentration of DHM was 1μM,and the treatment duration of co-culture was 24 hours.3.P2X₇ measurement results:The m RNA and protein expression levels of P2X₇in the HG+SP+CORT group were higher than those in the control group（p<0.01）.After treatment with DHM and P2X₇ sh RNA,the m RNA and protein expression levels of P2X₇ in the HG+SP+CORT+DHM and HG+SP+CORT+P2X₇sh RNA groups were significantly lower than those in the model group（p<0.01）.Immunofluorescence results showed that the expression levels of P2X₇ and GFAP in the HG+SP+CORT group were higher than those in the control group（p<0.01）.After DHM and P2X₇ sh RNA treatment,the co-expression levels of P2X₇ and GFAP in the HG+SP+CORT+DHM and HG+SP+CORT+P2X₇ sh RNA groups were significantly reduced（p<0.01）.4.IL-1βand TNF-αmeasurement results:The m RNA and protein expression levels of IL-1βand TNF-αin the HG+SP+CORT group were higher than those in the control group（p<0.01）.After treatment with DHM and P2X₇sh RNA,the m RNA and protein expression levels of IL-1βand TNF-αin the HG+SP+CORT+DHM and HG+SP+CORT+P2X₇ sh RNA groups were significantly reduced（p<0.01）.ELISA results showed that the expression levels of IL-1βand TNF-αin the HG+SP+CORT group were higher than those in the control group（p<0.01）.The expression levels of IL-1βand TNF-αin the DHM and P2X₇ sh RNA treatment groups were significantly lower than those in the HG+SP+CORT and HG+SP+CORT+scramble sh RNA group（p<0.01）.5.ERK 1/2 phosphorylation results:Western blot results showed that the IOD ratio of p-ERK 1/2 to ERK 1/2 in the HG+SP+CORT group was higher than the control group（p<0.01）,the IOD ratio of p-ERK 1/2 to ERK 1/2 in HG+SP+CORT+DHM and HG+SP+CORT+P2X₇ sh RNA group was significantly lower than that in HG+SP+CORT group（p<0.01）.6.Ca²⁺signal detection results:The Ca²⁺signal release of HG+SP+CORT group increased compared with the control group.After DHM and P2X₇ sh RNA treatment,the Ca²⁺signal was down-regulated to control levels（p<0.01）.7.Apoptosis results:Compared with the control group,we observed a significant increase in apoptosis in the HG+SP+CORT group（p<0.01）.The apoptosis after DHM and P2X₇ sh RNA treatment returned to normal levels（p<0.01）.8.Whole-cell patch-clamp experiment results:HEK293 cells expressing P2X₇receptor were activated by 100μM ATP,and DHM（100μM）significantly inhibited ATP activation current.Conclusion:DHM can protect primary hippocampal astrocytes co-cultured with HG+SP+CORT from P2X₇ receptor-mediated damage.P2X₇ receptor may become a new target for prevention and treatment of diabetic neuropathic pain and depression.DHM may be a potentially effective drug for the treatment of diabetic neuropathic pain with depression.HG+SP+CORT co-cultured cells may be a viable cell model for exploring diabetic neuropathic pain and depression cell damage.